Receptors Seminar No. 9 Q. 2 A. 2 • Allosteric protein (in membrane or cytosol) • It has two domains: • ligand-binding domain (with binding site for signal molecule) – changes conformations of receptor • effector domain – starts biological response to ligand (production of second messenger etc.) Q. 3 A. 3 Q. 4 A. 4 • signal molecule (ligand) - carries specific information into cell • has extremely low concentration in blood (10^-9 – 10^-15 mol/l) • binds to corresponding receptor • signal molecule is usually quickly inactivated • agonist – ligand which after binding to receptor transduces signal • antagonist – ligand which after binding to receptor blocks signal transduction TH no biological response Q. 5 Amplification of signal: 1 signal molecule 10 000-100 000 molecules of second messenger Q. 6 A. 6 Examples of second messengers [• ] Hydrophilic – cAMP, IP[3 ] • Lipophilic – diacylglycerol (DAG) • Inorganic – Ca^2+, NO Q. 9 A. 9 Two types of receptors: membrane and intracellular Three types of membrane receptors Ion channels in synapses, activated by neurotransmitters, very quick response Receptors activating G-proteins stimulate or inhibit adenylate cyclase /phospholipase C Receptors with enzyme activity guanylate cyclase - atrial natriuretic factors tyrosine kinase - insulin Q. 11 Acetylcholine formation / inactivation Acetylcholine formation Acetylcholine inactivation GABA formation / inactivation GABA formation GABA inactivation Q. 12 + 13 A. 12 + 13 • Excitatory neurotransmiters open cationic channels TH depolarization (more positive membrane potential) • Inhibitory neurotransmiters open anionic channels TH hyperpolarization (more negative potential) Nicotinic acetylcholine receptor ^• transmembrane protein = channel for Na^+ and K^+ • heteropentamer (α[2]βγδ) • α-subunits have two binding sites for acetylcholine (ACH) • nicotine (= xenobiotic) is agonist of this receptor Q. 15 – Four events on postsynaptic membrane and corresponding changes of membrane potential A. 15 Four events on postsynaptic membrane • ACH binds to receptor TH channel opens  influx of Na^+ and efflux of K^+ TH membr. potential changes (-60  -40 mV) • partial depolarization of membrane opens voltage-dependent Na^+-channel  further influx of Na^+  depolarization of postsyn. membrane ( +20 mV) • this depolarization opens K^+-channel (volt. dep.)  efflux of K^+  membrane potential returns to normal value (-60 mV) = repolarization • Na^+,K^+-ATPase gets ion distribution to normal state (Na^+  OUT, K^+  IN) GABA receptor • channel for chloride ion (Cl^-) • has the binding site for GABA TH channel opens  Cl^- ions get into cell  hyperpolarization ( -80 mV)  decrease of excitability • benzodiazepines and barbiturates (synthetic substances) have similar effects like GABA, they are used as anxiolytics and/or sedatives • endozepines – endogenous peptides have opposite effects, close the channel (are responsible for anxiety feelings) Diazepine Benzo[f]diazepine Benzodiazepines Barbiturates Receptors with adenylate cyclase system (Scheme on p. 4) Describe the pathway of signal G-Protein linked receptors • extracellular part of receptor has a binding site for hormone • intracellular part has a binding site for G-protein • G-proteins are heterotrimers (αβγ) • in resting state, α-unit has GDP attached • after binding hormone TH (α-GDP)βγ makes complex with receptor  GDP is phosphorylated to GTP • activated G-trimer dissociates: (α-GTP)βγ  α-GTP + βγ • α-GTP interacts with effector (enzyme)  activated/inhibited enzyme  second messenger (↑ or ↓) What reaction is catalyzed by adenylate (adenylyl) cyclase? Adenylyl cyclase reaction AMP is called also adenylic acid Adenylate (adenylyl) cyclase (AC) • membrane bound receptor [• ] catalyzes reaction: ATP ® cAMP + PP[i ]• G[s] protein stimulates AC  conc. of cAMP ↑ • G[i] protein inhibits AC  conc. of cAMP ↓ Q. 19 A. 19 • Protein kinase – phosphorylation by ATP Protein-OH + ATP  Protein-O-P + ADP • Protein phosphatase – hydrolysis of phosphate ester Protein-O-P + H[2]O  Protein-O-H + P[i ] General scheme of phosphorylation Three amino acids have a hydroxyl group in the side chain • Serine (3 C, primary alcohol hydroxyl) • Threonine (4 C, secondary alcohol hydroxyl) • Tyrosine (3 + 6 C, phenolic hydroxyl) Phosphatidyl inositol system (Scheme on p. 4) The structure of PIP[2 ]Q. What is the source of inositol in human body? The origin of inositol Main types of G-proteins Q. 20 A. 20 Receptors with guanylate cyclase • second messenger = cGMP • activates protein kinase G (PKG) A. 23 • By the action of NO • In smooth muscle cells (chapter 14) Insulin receptor • has four subunits (α[2]β[2]) • extracellular α-units bind insulin • intracellular β-units have tyrosine kinase activity TH phosphorylation of tyrosine phenolic hydroxyl of intracellular proteins including insulin receptor itself (autophosphorylation)  cascade of further events  biological response Intracellular receptors: - cytoplasmatic - nuclear for non-polar signal molecules steroids, iodothyronines, calcitriol, retinoids Intracellular receptors Q. 30 A. 30 • HRE = hormone response elements • regulatory DNA sequences, bind complexes of hydrophobic hormones with their intracellular receptors • They act as enhancers or silencers • Located at the beginning of regulatory DNA region • 5’-----HRE ---Promoter-------3’ Steroid and thyroid hormones • insoluble in water TH in ECF are transported in complex with transport proteins • hormone themselves diffuse easily across cell membrane • they are bound to cytoplasmatic or nuclear receptors • in nucleus, the hormone-receptor complex binds to HRE (hormone response element) in regulation sequence of DNA • this leads to induction of mRNA synthesis = transcription of gene Cholinergic synapses • neurotransmitter: acetylcholine • two types of receptors • nicotinic rec. (ion channel) – e.g. neuromuscular junction • muscarinic rec. (G-prot.) – e.g. smooth muscles Cholinergic receptors Q. 31 A. 31 • Presynaptic membrane contains voltage-gated calcium channels • influx of Ca^2+ activates protein kinase which phosphorylates synapsin and other proteins • this triggers the fusion of presynaptic vesicles (contaning acetylcholine) with cell membrane and exocytosis of acetylcholine • acetylcholine is liberated into synapse Q. 32 A. 32 Q. 33 What is nicotine? Nicotine is the main alkaloid of tobacco (Nicotiana tabacum) Q. 34 What is muscarine? A. 34 Muscarine is quaternary ammonium alkaloid in some mushrooms Q. 35 A. 35 • nicotine binds to acetylcholine nicotinic receptors in the brain and other tissues including cells of adrenal medulla • activation of nicotinic receptor TH change of membrane potential  exocytosis of vesicles with adrenaline  secretion of adrenaline TH metabolic processes typical for acute stress (see Seminar No. 6) Other effects of nicotine: • increases the secretion of saliva and gastric juice • increase intestinal peristalsis • vasoconstriction Inhibitors of acetylcholinesterase • Reversible – carbamates (N-substituted esters of carbamic acid), e.g. fysostigmine, neostigmine • they are used to improve muscle tone in people with myasthenia gravis and routinely in anesthesia at the end of an operation to reverse the effects of non-depolarising muscle relaxants. It can also be used for urinary retention resulting from general anaesthetia • Irreversible – organophosphates, very toxic compounds (chemical warfare agents) Carbamates – General formulas Carbamates – General formulas Organophosphates Q. 36 Adrenergic synapses • neurotransmitter: noradrenaline [• ] four types of receptors: α[1], α[2], β[1], β[2 ]• all of them are G-protein linked receptors • occur in various cells and tissues Adrenergic receptors Q. 37 Describe the synthesis of noradrenaline. The formation of DOPA and dopamine Noradrenaline and adrenaline